The SPR biosensor is a sensitive real-time technique, which can be used to extract information about molecular interaction near certain metal surfaces. It offers the possibility to determine concentration, association and dissociation rate constants and affinity as well as epitope mapping and determination of interaction specificity [B. Liedberg and K. Johansen, Affinity biosensing based on surface plasmon detection in “Methods in Biotechnology, Vol. 7: Affinity Biosensors: Techniques and Protocols”, K. R. Rogers and A. Muchandani (Eds.), Humana Press Inc., Totowa, N.J., pp. 31-53]. One of the components participating in the studied reaction is immobilized on the metal surface either before or during the SPR experiment. The immobilized molecule is exposed to a continuous flow into which one can inject interacting species. The method is based on optical detection and the sensing signal reflects changes in dielectric function or refractive index at the surface. These changes can be caused by molecular interaction at the surface.
The PCM technique is based on an oscillating piezoelectric crystal in a microbalance device, wherein the crystal consists of e.g. quartz, aluminum nitride (AlN) or sodium potassium niobiates (NKN. When the crystal is a quarts crystal, the device is referred to as a QCM (quartz crystal microbalance). The PCM and QCM are gravimetrical sensors and are thus sensitive to mass changes. A QCM comprises a piezoelectric quartz crystal plate upon which metal electrodes have been deposited on both sides. An alternating potential difference applied on such a crystal plate induces shear waves. At certain frequencies—such that the thickness is an odd integer of half wavelengths—the crystal will be in resonance [M. Rodahl, F. Höök, A. Krozer, P. Brzezinski and B. Kasemo, Quartz crystal microbalance setup for frequency and Q-factor measurements in gaseous and liquid environments, Review of Scientific Instruments 66 (1995) pp. 3924-3930] and [Saurbrey,Z.Phys. (1959), pp155, 206-222]. The energetically most favourable number of half wavelengths is one. The resonance frequency is dependent on the thickness of the crystal, but is normally in the MHz range. A mass change on the surface of the plate will result in a shift in the resonance frequency. The fact that frequency shifts of 0.01 Hz can be easily measured makes the QCM a sensitive sensor for determining mass variations. A number of patents and other publications describe the use of piezoelectric quartz crystals (QCM) as affinity-based chemical sensors/detectors in e.g. various immuno-assay techniques, and detection of bacteria and virus. In most of these applications the QCM-instrument is used to analyze the weight gain of the crystal after interaction between antibodies and antigens.
The crystal is used as a microbalance to measure very small masses. The thin piezoelectric sensor crystal electrode used in our experiments has gold evaporated on each side. The crystals can be made to oscillate at its resonance frequency by applying an AC-voltage over the electrode. The principle behind the QCM-technique is that the resonant frequency changes when the mass of the crystal changes. By using this method the mass changes in a bio-molecule layer of a crystal can be monitored. Many studies have been reported utilizing the QCM, where the crystal has been coated with a coating that interacts in a specific way with a molecule or particle, e.g. a bacterium, virus, antibody or antigen, resulting in a loss or gain of weight of the crystal, which change in weight is measured.
There are obvious difficulties in analyzing small molecules with conventional immunosensors due to the low response, i.e. small change in weight of the sensor crystal. For attaining the necessary detection of small molecules, the sensitivity of the system has to be improved. This may be achieved by using displacement reactions where a large antibody molecule is detached from the sensor surface by dissociation and reaction with an analyte antigen that has a higher affinity to the antibody than the antigen bound to the sensor surface.(Willner et. al EP 0 843 816).